Marie-Josée Gagné

Detection of Human Food-Borne and Zoonotic Viruses on Irrigated, Field-Grown Strawberries

ABSTRACT This study evaluated the presence of pathogenic human and zoonotic viruses on irrigated, field-grown strawberries. Norovirus genogroup I, rotavirus, and swine hepatitis E virus genogroup 3 were detected on strawberries, and irrigation water is suspected as the contamination origin.

The feline calicivirus as a sample process control for the detection of food and waterborne RNA viruses.

Many food and waterborne outbreaks of infectious disease are caused by viruses. While numerous methods exist and are being developed to test food and water for the presence of enteric viruses, there is no standard control for the comparison of different methods. Potential control viruses should be well characterized, share the physical characteristics of the enterically infecting viruses and not normally be associated with foods. Here, the feline calicivirus (FCV) is proposed as a sample process control for methods aimed at the extraction and detection of RNA viruses in food and water. FCV is shown to be useful as a control for the extraction of hepatitis A virus (HAV) from water using filtration technology and from strawberries using the Pathatrix system. The FCV standard provides a valuable quality control tool when testing potentially contaminated food samples.

Hepatitis E virus load in swine organs and tissues at slaughterhouse determined by real-time RT-PCR

Although uncommon in North America, Hepatitis E virus (HEV) has been identified in some industrialized countries in patients without a history of travel to HEV-endemic countries. Its presence is ubiquitous worldwide in swine populations. Zoonotic transmission of swine HEV to non human primates has been achieved experimentally and transmission of HEV after ingestion of contaminated raw or undercooked meat is well documented. In Canada, so far, no HEV outbreak has been documented but HEV genotype 3 strains have been identified in sera and faecal samples of swine origin. The objective of the present study was to determine the viral load of HEV in liver, loin, bladder, hepatic lymph node, bile, tonsil, plasma and faeces samples of 43 pigs at slaughter. Feline calicivirus (FCV) was used as sample process control to validate the RNA extraction process, as a confirmation of the absence of sample inhibitors and as an amplification control. Using FCV/HEV multiplex TaqMan RT-qPCR system, HEV RNA was detected in 14 out of the 43 animals tested. HEV was detected in lymph nodes (11/43), bladder (10/43), liver (9/43), bile (8/43), faeces (6/43), tonsils (3/43), plasma (1/43) samples from infected animals. No HEV-positive loin samples were observed. Viral loads of 10(3) to 10(7) copies/g were estimated in positive liver and bile samples.

Development of a real‐time TaqMan PCR assay for the detection of porcine and bovine Torque teno virus

Aims:  The goal of this study was to develop and to optimize molecular tools to detect the presence of Torque teno virus (TTV) in swine and cattle. A novel real‐time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect both genogroups of TTV strains.

Torque Teno Virus in Children Who Underwent Orthotopic Liver Transplantation: New Insights About a Common Pathogen

BACKGROUND  Torque Teno virus (TTV) is a ubiquitous infectious agent. Transplant recipients are at risk of hepatitis E virus (HEV) infection and could be vulnerable to TTV-associated adverse effects. The aim of this study was to evaluate the influence of immunosuppression and HEV infection on TTV replication and liver injury in pediatric patients after orthotopic liver transplantation (OLT). METHODS  Pediatric recipients of liver transplants were classified into the following 2 groups: (1) those with normal serum aminotransferases levels and (2) those with persistently increased serum aminotransferases levels and histological features of chronic hepatitis of unknown etiology. The TTV load was assessed in 342 serum samples by use of TaqMan real-time polymerase chain reaction, along with TTV genogroups and coinfection with HEV. RESULTS  TTV DNA was detected in 96% of tested serum samples. Viral load was significantly lower in patients with features of chronic hepatitis, of whom 78% had liver fibrosis scores of ≥2. Viral load decreased during posttransplantation follow-up. Viral load and genogroups were influenced by immunosuppression. Lower viral load was observed in patients coinfected with HEV. CONCLUSIONS  TTV infection is widespread, and its replication is closely related to immune status and viral coinfection. High TTV viremia is not associated with hepatitis after OLT, but, conversely, liver inflammatory activity impairs TTV replication.

Simultaneous recovery of bacteria and viruses from contaminated water and spinach by a filtration method.

Water and leafy vegetables eaten fresh are increasingly reported as being involved in food-borne illness cases. The pathogenic agents responsible for these infections are mainly bacteria and viruses and are present in very small quantities on the contaminated food matrices. Laboratory techniques used to isolate or detect the contaminating agent differ enormously according to the type of microorganisms, generating time and economical losses. The purpose of this study was to optimize a single method which allows at the same time the recovery and concentration of these two main types of pathogenic organisms. Water and spinach samples were artificially contaminated with the feline calicivirus (FCV), rotavirus, hepatitis A virus (HAV), Escherichia coli, Listeria monocytogenes, Campylobacter jejuni and Salmonella Typhimurium. The principle behind the recovery technique is based on the use of a positively charged membrane which adsorbs both viruses and bacteria present in the water or in the rinse from the vegetables. Using conventional microbiology, PCR and RT-PCR, this filtration technique allowed a detection level superior to 10² CFU/g for S. Typhimurium, E. coli, L. monocytogenes and C. jejuni and to 10¹ PFU/g for FCV, HAV and rotavirus. This combined method can also be applied to other bacterial and viral species for the identification of the responsible agent for food-borne illnesses.

Characterization of novel porcine sapoviruses.

Sapoviruses are common caliciviruses known to cause enteric diseases in humans and animals. SaVs are genetically highly heterogeneous and are presently classified in five genogroups that are further subdivided in a number of genotypes. In recent years, a number of novel animal SaV strains, mostly of swine origin, have been partially characterized and proposed to represent novel genogroups or genotypes. We previously reported the detection and partial characterization of a wide range of variable and novel SaV strains of uncertain taxonomic status in Canadian swine. We now report on further genomic characterization of two novel strains to clarify their taxonomic relationship to other swine and human SaVs. Detailed analysis of different regions of their genomes, including determination of their complete capsid sequence, did not permit clear taxonomic assignment according to current criteria. This situation appears reminiscent of that of a number of SaV strains of swine origin and calls for a classification update for this calicivirus genus. We also report the detection of swine GIII SaVs for the first time in Canada.

The effect of pre-treatment and sonication of centrifugal ultrafiltration devices on virus recovery

Enteric viruses such as norovirus (NV) and hepatitis A (HAV) are responsible for a large proportion of food and water-borne illnesses. Most human pathogenic enteric viruses cannot be cultured so they must be detected by molecular techniques. Male specific (F(+)) RNA coliphages, a potential surrogate for human enteric viruses, can be detected by culture and molecular assays. Numbers of viruses and F-RNA coliphages in contaminated food or water may be too low for direct detection. Ultrafiltration is a general concentration method for all virus types but there is little information on the recovery efficiency of F-RNA coliphages and enteric viruses. The recovery of F-RNA coliphage MS2 was only 25% by plaque assay in initial trials. The objective was to optimize the recovery of concentrated MS2 from Microsep 100K ultrafiltration devices. The mean recovery of MS2 increased significantly to 85% by plaque assay and 65% by real-time RT-PCR when ultrafiltration devices were treated with 1% BSA before concentration and then ultrasonicated after concentration. The method was validated with MS2, HAV, NV and feline calicivirus (FCV) in water and spinach eluate. The recovery of MS2, HAV and NV was significantly higher from concentrates obtained from water with treated devices than untreated devices but not significantly different for FCV or from spinach eluate. To our knowledge, this is the first study to use ultrasonication as a post-treatment step to increase recovery of viruses from ultrafiltration devices.

Evaluation of survival of murine norovirus-1 during sauerkraut fermentation and storage under standard and low-sodium conditions

Sodium reduction strategies have raised a few concerns in regards to possible outbreaks in unpasteurised raw fermented vegetables. Among potential outbreak agents, foodborne viruses are recognized as an important cause of food-borne illnesses. As most of them are acid-resistant, evaluation of the efficacy of lactic fermentation in inactivating enteric viruses must be considered to ensure the safety of these foods. In particular with the sodium reduction trend which could impair adequate fermentation in vegetables, we have challenged sauerkraut fermentation at a final concentration of 4 log TCID50/mL with the murine norovirus (MNV-1). Three sodium chloride concentrations (1.0%, 1.5%, 2.0%) were evaluated in spontaneous and starter fermentation of sauerkraut and were followed during fermentation and over a storage phase of 90 days. Detection of MNV-1 genetic material was carried out by real-time RT-PCR and the infectivity on cell culture. Real-time RT-PCR results showed that viral RNA was still detected after 90 day in sauerkraut under all the different conditions. Furthermore, MNV-1 viral particles were able to infect RAW cells after 90 days of storage with a non-significant viral charge reduction. Sodium reduction has a significant impact on the fermentation processing of sauerkraut but no influence on the destruction of norovirus particles or on their survival.

Presence, viral load and characterization of Torque teno sus viruses in liver and pork chop samples at retail

Torque teno viruses (TTV) are widespread in humans, swine as well as in several other animal species. In market ready swine, the reported prevalence ranges between 11% and 100%. Through a national retail sampling plan from the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) program, 283 and 599 liver and pork chop samples, respectively, were collected over a 12-month period from commercial establishments in 5 selected geographical regions of Canada to assess the presence of Torque teno sus viruses (TTSuVs) in these products. TTSuVs were detected in 97.9% of pork chops with viral loads ranging between 1×10(4) and 9.9×10(5) genomic copies (gc)/g and 98.6% of liver samples with viral loads ranging from 1×10(5) to 9.9×10(6) gc/g. A selection of 20 positive samples (10 pork chop and 10 liver) from the 5 geographical regions were further tested for the production, of a 305bp fragment for TTSuV1 and a 253bp fragment for TTSuV2 in the non-coding region. TTSuV1 was present in all 10 liver and 10 pork chops samples while TTSuV2 was detected in 10 liver and 9 pork chop samples. Two different TTSuV1 sequences were simultaneously detected from 5 of 20 samples and 2 different TTSuV2 sequences were detected from 6 of 19 samples. The omnipresence of TTSuVs in commercial pork samples may allow its use as a viral indicator to monitor the effectiveness of cleaning and disinfecting process in slaughtering, cutting, slicing and packaging facilities.